Translation in all organisms is performed by the ribosome, one of the most ancient and universally conserved molecular machines. Termination is the last step of protein synthesis, which ensures that expressed proteins have lengths strictly defined by the corresponding open reading frames. This critical step should be accurate and efficient to prevent accumulation of truncated or overly long polypeptide products that can be toxic to cell. Whereas most steps of translation and corresponding extraribosomal factors (e.g. elongation factors) are highly conserved between bacteria and eukaryotes, the termination of translation is not. In human, premature termination is associated with a large number of genetic diseases. These unique features place termination as a promising target for development of drugs. A detailed understanding of bacterial translation termination may provide us with tools to develop antibacterial drugs. Elucidation of the molecular mechanism of eukaryotic termination is necessary to design or search for therapeutics to target human neurological diseases linked to premature termination. In this proposal, we will gain insights into molecular mechanisms of termination in both bacteria and eukaryotes. In Aim 1, we will determine crystal structures of intermediate conformational steps of termination on the bacterial 70S ribosome. This will provide insights into how a remarkable accuracy is achieved by bacterial release factors RF1 and RF2. In Aim 2, we will study termination on the eukaryotic 80S ribosome. Here, essential class I release factor eRF1 and class II release factor eRF3 are involved, which are structurally distinct from their bacterial counterparts. In Aim 3, we will explore new therapeutic routes against diseases caused by premature termination. The long-term goals of our studies are to gain detailed mechanistic insights into key cellular processes involving translation, and to contribute to therapeutics development.